A modified tetracycline-regulated system provides autoregulatory, inducible gene expression in cultured cells and transgenic mice.

Abstract

A system for tetracycline-regulated inducible gene expression was described recently which relies on constitutive expression of a tetracycline-controlled transactivator (tTA) fusion protein combining the tetracycline repressor and the transcriptional activation domain of VP16 [Gossen, M. & Bujard, H. (1992) Proc. Nati. Acad. Sci. USA 89, [5547][5548][5549][5550][5551]. This system yielded only low levels of transactivator protein, probably because tTA is toxic. To avoid this difficulty, we placed the tTA gene under the control of the inducible promoter to which tTA binds, making expression of tTA itself inducible and autoregulatory. When used to drive expression of the recombination activating genes 1 and 2 (RAG-1 and RAG-2), the autoregulatory system yielded both substantially higher levels of variable (diversity) joining [V(D)J] recombination activity (70-fold on average) and inducible expression in a much larger fraction of transfected cells (autoregulatory, 90%, vs. constitutive, 18%). In addition, this system allowed the creation of transgenic mice in which expression of a luciferase transgene was inducible tens to hundreds of times the basal levels in most tissues examined. Induced levels of expression were highest in thymus and lung and appear to be substantially higher than in previously reported inducible luciferase transgenic mice created with the constitutive system. With the modified system, inducible transactivator mRNA and protein were easily detected in cell lines by RNA and Western blotting, and transactivator mRNA was detected by RNA blotting in some tissues of transgenic mice. This autoregulatory system represents an improved strategy for tetracycline-regulated gene expression both in cultured cells and in transgenic animals.

Induction of erythroid differentiation by inhibition of Ras/ERK pathway in a Friend murine leukemia cell line

“…Using this cell line, we established two subclones which express either an activated (12V) or a dominant-negative (17N) mutant of H-ras oncogene (Feig and Cooper, 1988;Campbell et al, 1998) by using a tetracycline (Tet)-regulated expression system (Tet-o system) (Gossen and Bujard, 1992;Shockett et al, 1995). In these clones (named ras17N/SKTtet and ras12V/SKTtet, respectively), strong induction of the transgene expression was observed both at the levels of mRNA and protein after withdrawal of Tet from the medium (Figure 1a).…”

Expression of Bcl-XS alters cytokinetics and decreases clonogenic survival in K12 rat colon carcinoma cells

“…This system also possesses an auto-regulatory feedback loop (neither the tTA nor the transgene is produced at signi®cant levels unless tetracycline is absent), eliminating possible artifacts (such as transcriptional squelching) from constitutive tTA expression (Iida et al, 1996). Lastly, the system is introduced into target cells via a retrovirus allowing transduction of cells that are inherently di cult to transfect or for use in vivo to assess novel gene therapy protocols in a regulated fashion (Dhawan et al, 1995;Iida et al, 1996;Furth et al, 1994;Shockett et al, 1995). Drawbacks of this system include the heterogeneous expression seen in Figure 9.…”

Translating Lung Function Genome-Wide Association Study (GWAS) Findings

“…Inducible knock out mice can also be produced whereby gene expression can be turned on or off by doxycycline or tetracycline-regulated systems (Gunther et al, 2002;Shockett, Difilippantonio, Hellman, & Schatz, 1995). A way of producing lung specific inducible knockout mice is by exploiting the cell make-up of the airways, i.e.…”